PROJECT SUMMARY We recently demonstrated that ubiquitin-specific protease-20 (USP20) is scaffolded by the adaptor protein known as ?-arrestin2 (?arr2), and that USP20 desensitizes ubiquitin-dependent signaling from Toll-like receptor-4 (TLR4) to NF?B activation by deubiquitinating TRAF6 and ?arr2. Using transgenic mice expressing dominant-negative USP20 in smooth muscle cells, we found that USP20 reduces neointimal hyperplasia after arterial injury and that USP20 activity in SMCs reduces atherosclerosis in Ldlr-/- mice. To establish anti-atherogenic effects of systemically expressed USP20, and to elucidate further molecular mechanisms by which USP20 protects against atherosclerosis, this project will test the hypothesis that USP20 attenuates atherosclerosis by deubiquitinating several substrate proteins that were previously unassociated with USP20 but that are important in signaling pathways that activate NF?B: ?arr1, TRAF6, TRAF2, and RIPK1. Furthermore, because USP20 employs ?arr2 as a scaffold to facilitate association with distinct proteins, and because ?arr1 reduces vascular inflammation, this project will test whether USP20?s anti-atherogenic activity involves ?arr1-mediated scaffolding. To these ends, this project will study systemic effects of USP20 on atherosclerosis by comparing Usp20-/- /Ldlr-/- versus Ldlr-/- mice, on a background of ?arr1+/+ or ?arr1-/+. To determine the effects of endothelial USP20 on atherosclerosis, this project will compare atherosclerosis among VECad-Cre- ERT2/Usp20flox/flox/Ldlr-/- vs. Usp20flox/flox/Ldlr-/- mice treated tamoxifen; furthermore, we will investigate cytokine secretion, and dynamic ubiquitination of signaling proteins in primary aortic endothelial cells that are WT, Usp20-/-, Usp20-/-/?arr1-/+, or ?arr1-/-. To determine what kinase in endothelial cells phosphorylates USP20 on Ser333 (and thereby abrogates USP20 deubiquitinase activity), this project will test IRAK1, PAK1, and ROCK1 with several loss-of function approaches, including a USP20 minigene, in primary endothelial cells. These studies collectively may identify USP20 phosphorylation as novel therapeutic target for atherosclerosis.